This project will employ genetic and molecular approaches to analyze the structure and function of introns and the mechanism of RNA splicing using yeast mitochondria as the experimental system. Sequence analysis of existing and new cis-acting splicing defective mutations in introns 4 and 5 of the cytochrome b (cob) gene will be employed to define those intron sequences that serve as portions of the splicing substrate. The role of those sequences in splicing reactions will be assessed by characterizing partial revertants in experiments especially designed to learn the role of secondary and higher order structures in the splicing of those introns. The positions of splice junctions of those introns (and others) will be determined by cDNA sequencing and such sequences will be compared to learn whether one or more consensus sequences exist in this system. It already appears that such common splice junction sequences found in nuclear systems are different from those in this system. We will refine our current model of the expression of trans-acting splicing factors encoded by cob intron 4 by testing several submodels explicitly. We will also test the generality of the concept that introns which contain open reading frames encode intron specific splicing factors (maturases) by analyzing the four translatable introns of the oxi3 gene. Nuclear genes whose products participate in mitochondrial RNA splicing will be identified by the isolation of nuclear suppressors of mitochondrial splicing defective mutants. Those suppressors will be used to identify null alleles at the same loci; that is, we will use a novel genetic screening method to isolate nuclear mutants defective in splicing mitochondrial introns. Such mutants should be very useful in future attempts to study the RNA splicing apparatus in detail. These studies should lay a solid foundation for detailed molecular studies of the mechanism of RNA splicing in yeast mitochondria.